Duplication of the human genome depends on the activation of thousands of initiation sites where DNA synthesis is programmed to start. However, in spite of great advances in the field it is not clear what the distribution is of these sites along each one of the chromosomes, nor whether this distribution changes with the physiological state of the cell. Our long-term goal is to obtain a detailed map of these sites along human chromosomes to deduce initiation signatures that may allow us to distinguish normal from abnormally growing cells, thus providing us with a tool to detect early stages of abnormal cell proliferation. Based on a PCR-based nascent strand abundance assay on a 99 kb region of chromosome 2, we have obtained preliminary data suggesting that cancer cells differ from their normal counterparts in the distribution of initiation sites. In the present proposal, we wish to use DNA microarray technology to expand these studies to longer chromosome regions that are relevant in breast cancer. We hypothesize that the frequency, distribution, and temporal activation of initiation sites in these regions is different in cancer cell lines compared to their normal counterparts. To test this hypothesis, our specific aims are: (1) to map and compare the location of initiation sites on breast cancer-relevant regions of chromosomes 3, 17 and 20, in both normal and breast cancer cell lines. To accomplish this objective we plan to (a) isolate short (about 1-1.5 kb) nascent DNA strands from asynchronously growing cells;(b) quantitate their abundance on DNA tiling arrays containing 60-mer probes covering a 20 Mb region on Chr20q12-13, two 4Mb regions on Chr17p13 and Chr17q23, respectively, and a 3 Mb region on Chr3q26;and (c) compare the profiles of normal and cancer cell lines. (2) To assess the temporal order of activation of initiation sites along cancer-relevant regions of chromosomes 3, 17 and 20 in both normal and cancer breast cell lines. To this end we plan to synchronize cells in G1/G0 and probe the tiling path DNA microarray described above, with short nascent DNA strands obtained at different time points after entrance into the S phase. These studies will provide us with novel information about the initiation and temporal activation of DNA replication in both normal and malignant cells which will help identify tumor-specific initiation sites. This information will also offer new approaches for the diagnosis and control of abnormal cell growth.